Fermentative production of oligosaccharides by total fermentation utilizing a mixed feedstock

ABSTRACT

Disclosed are genetically engineered microbial cells for the production of oligosaccharides comprising a galactose-β1,4-glucose moiety at their reducing end, wherein said microbial cells are able to produce said oligosaccharides in the absence of exogenously added lactose, and a method of producing said oligosaccharides using said microbial cells.

The present invention relates to bacterial host cells being capable toproduce lactose or an oligosaccharide of interest which comprises agalactose-β1,4-glucose moiety at its reducing end, as well as to amethod of producing lactose or an oligosaccharide of interest whichcomprises a terminal galactose-β1,4-glucose moiety.

BACKGROUND

Human milk comprises a complex mixture of carbohydrates, fats, proteins,vitamins, minerals and trace elements. The most predominant fraction ofhuman milk consists of carbohydrates. The fraction of carbohydrateswithin human milk can be further divided into (i) lactose and (ii)oligosaccharides (human milk oligosaccharides, HMOs). Whereas lactose(galactose-β1,4-glucose) is used as an energy source, theoligosaccharides are not metabolized by the infant. The fraction ofoligosaccharides accounts for up to 1/10 of the total carbohydratefraction and consists of probably more than 150 differentoligosaccharides. The occurrence and concentration of these complexoligosaccharides are specific to humans and thus cannot be found inlarge quantities in the milk of other mammals including dairy farmanimals.

The most prominent human milk oligosaccharides are 2′-fucosyllactose and3′-fucosyllactose which together can contribute up to ⅓ of the total HMOfraction. Further prominent HMOs present in human milk arelacto-N-tetraose, lacto-N-neotetraose and the lacto-N-fucopentaose I.Besides these neutral oligosaccharides, also acidic HMOs can be found inhuman milk such as 3′-sialyllactose, 6′-sialyllactose and3-fucosyl-3′-sialyllactose, sialyl-lacto-N-tetraose,disialyl-lacto-N-tetraose. Notably, the vast majority of HMOs comprise agalactose-β1,4-glucose moiety at their reducing end. The structures ofthe HMOs are closely related to epitopes of epithelial cell surfaceglycoconjugates, the Lewis histoblood group antigens such as Lewis x(LeX). The structural similarity of HMOs to epithelial epitopes accountsfor the HMOs protective properties against bacterial pathogens.

The presence of oligosaccharides in human milk is known for a long timeand the physiological functions of these oligosaccharides were subjectto medical research for many decades. For some of the more abundanthuman milk oligosaccharides, specific functions have already beenidentified.

Besides the local effects in the intestinal tract as mentioned hereinbefore, HMOs have also been shown to elicit systemic effects in infantsby entering their systemic circulation. Also, the impact of HMOs onprotein-carbohydrate interactions, e.g. selectin-leukocyte binding, canmodulate immune responses and reduce inflammatory responses. Inaddition, it becomes more and more recognized that HMOs represent a keysubstrate for the development of infants' microbiomes.

Due to the well-studied beneficial properties of prebioticoligosaccharides, in particular of HMOs, but their limited availabilityfrom natural sources, an efficient commercial, i.e. large-scale,production of HMOs is highly desirable.

Attempting for large scale production of individual human milkoligosaccharides, chemical routes to some of these oligosaccharides weredeveloped. However, such methods involve the use of several noxiouschemicals, which impose the risk to contaminate the final product. Atleast large-scale quantities as well as qualities sufficient for foodapplications cannot be provided until today through chemical synthesis.

To bypass the drawbacks associated with the chemical synthesis of humanmilk oligosaccharides, several enzymatic methods and fermentativeapproaches for their production were developed. Fermentative productionprocesses have been developed for several HMOs such as2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose,lacto-N-neotetraose, 3′-sialyllactose and 6′-sialyllactose. Theseproduction processes typically use genetically engineered bacterialstrains such as recombinant Escherichia coli.

Today, all fermentative production processes as well as biocatalyticreactions to produce HMOs are based exclusively on exogenously addedlactose as the starting substrate. One or more monosaccharides are addedto lactose in the processes (U.S. Pat. No. 7,521,212 B1; Albermann etal., (2001) Carbohydr. Res. 334(2) p 97-103). The addition ofmonosaccharides to lactose can either be catalyzed byglycosyltransferases or by glycosidases using suitable activatedmonosaccharide substrates. In addition, additional monosaccharides canbe added to lactose by transglucosidase reactions.

In particular the fermentative production of HMOs proved to beefficient, because the nucleotide activated monosaccharides that arerequired but difficult to synthesize are provided by the metabolism ofthe microbial cells that are employed. However, the use of whole cellsfor the synthesis of HMOs also bears—in comparison to the biocatalyticapproach—several major disadvantages, which relate to transportprocesses across the cell membrane, to metabolic side reactions and tothe necessity that the oligosaccharides being synthesized by themicrobial cells have to be purified from a complex mixture containing,inter alia, various polyols (e.g. carbohydrates), nucleic acids,polypeptides, inorganic material, etc.

A technical problem related to the use of lactose in fermentativeprocesses that needs to be overcome, in particular when theoligosaccharide to be produced shall be used in human consumption, isthe rearrangement of lactose (beta-D-galacto-pyranosyl-(1->4)-D-glucose)into lactulose (beta-D-galactopyranosyl-(1->4)-D-fructofuranose) uponthermal treatment of lactose. This rearrangement can occur extensivelyby heat sterilization of lactose, leading to the rearrangement ofseveral percent of the lactose to be present in the fermentation mediumor the lactose fermentation feed to lactulose. However, lactulose is anon-digestible sugar for humans and is widely used as laxative in thetreatment of chronic constipation.

The conversion of lactose to lactulose not only leads to the generationof undesired lactulose, but also provides an undesired substrate forglycosylation reactions in the microbial cells. Thereby, more complexoligosaccharides (e.g. 2′-fucosyl-lactulose) are generated asby-products. Thus, the generation of lactulose from lactose is leadingto the contamination of the desired product with closely relatedoligosaccharides, which are difficult or even impossible to separatefrom the desired product.

Furthermore, lactose can be converted to allolactose(beta-D-galactopyranosyl-(1→6)-D-glucopyranose), another unwantedcontaminant (Huber et al., “Efflux of beta-galactosidase products fromEscherichia coli” (1980) J. Bacteriol. 141, 528-533), if supplied to abeta-galactosidase positive E. coli strain.

Moreover, the addition of lactose may cause a well-documented effectknown as “lactose induced cell killing”. This effect is most likelycaused by the excessive uptake of lactose by the microbial cell and theassociated collapse of the proton gradient across the bacterialmembrane. In particular the overexpression of the lactose permease gene(e.g. lacY of E. coli) in combination with the exposure of therecombinant microbial cell to excess lactose can cause a considerablegrowth delay of the recombinant strain and an increased synthesis ofcellular polysaccharides (Grube et al., “Hydrogen-producing Escherichiacoli strains overexpressing lactose permease: FT-IR analysis of thelactose-induced stress” (2013) Biotechnol. Appl. Biochem. 5, 31).

Furthermore, any commercially available lactose today is derived fromwhey, a waste product of the dairy industry. Whey is produced inenormous quantities in cheese and casein manufacturing. Thus, beingderived from the dairy industry there are still concerns related to apotential contamination of lactose with prion proteins which are thecausative agent of bovine spongiform encephalopathy (BSE), also widelyknown as mad cow disease. BSE is a fatal neurodegenerative disease incattle, causing spongy degeneration of the brain and spinal cord. BSEcan be trans-mitted to humans and is there known as variant ofCreutzfeldt-Jakob disease.

Above all, lactose is still one of the most expensive components of thefermentation medium and its substitution by glucose, glycerol, sucroseetc. would lead to a more cost-efficient production of HMOs.

To overcome aforementioned drawbacks, improved means and methods for theproduction of HMOs were developed. For example, WO 2015/150328 A1discloses bacterial host cells being capable to produce oligosaccharidescomprising a terminal galactose-(1→4)-glucose disaccharide, wherein saidbacterial host cell expresses at least one recombinant nucleic acidsequence encoding for a β-1,4-galactosyltransferase which is able togalactosylate a free glucose monosaccharide to intracellularly generatelactose, and which contains and expresses at least one recombinantnucleic acid sequence encoding a fucosyltransferase, asialyltransferase, a glucosamyltransferase or a galactosyltransferase.Said bacterial host cell is capable of generating the oligosaccharidewithout exogenous addition of lactose such that the bacterial host cellcan be cultivated in a culture medium without exogenous addition oflactose to produce said oligosaccharide. More specifically, WO2015/150328 A1 discloses a genetically engineered E. coli strain for theproduction of 2′-fucosyllactose which utilizes sucrose or a combinationof glucose and sucrose as carbon source. For utilization of sucrose,said E. coli strain was genetically engineered to expresses the fourgenes of the csc-gene cluster of E. coli W, i.e. the genes encoding thesucrose permease (cscB), the fructokinase (cscK), the sucrose hydrolase(cscA), and a transcriptional repressor (cscR).

However, producing 2′-FL by said genetically engineered E. coli strainusing sucrose as sole carbon and energy source has its drawbacks in thatit is also difficult to heat sterilize sucrose without a considerabledegree of hydrolyzation and formation of unwanted side products. As analternative sterile filtration of sucrose solution can be employed, butthe sterile filtration bears a high risk of foreign growth contaminationof the fermentation in particular in industrial-scale fermentation.

In addition, cultivating a microbial cell for the production of an HMOin the presence of sucrose as carbon source, wherein said microbial cellhas been genetically engineered to possess a split metabolism such thatthe monomers constituting sucrose are used in distinct metabolicpathways, leads to undesired growth characteristics of the bacterialcell culture, presumably due to the stoichiometry of monomers generatedfrom intracellular sucrose hydrolyzation which does not match thequantitative needs of the different monomers in the distinct pathways.

To overcome aforementioned drawbacks, a genetically engineered microbialcell is provided which is capable of producing an oligosaccharide ofinterest comprising a galactose-β1,4-glucose moiety at its reducing endwhen cultivated on a mixed monosaccharide feedstock as main carbon andenergy source but in the absence of exogenously added lactose.

SUMMARY

In a first aspect, provided is a genetically engineered microbial cellfor the production of lactose or an oligosaccharide of interestcomprising a galactose-β1,4-glucose moiety at its reducing end, whereinsaid microbial cell is capable of producing said lactose de novo or saidoligosaccharide of interest when cultivated in the absence ofexogenously added lactose.

In a second aspect, provided is the use of the genetically engineeredmicrobial host cell for the production of lactose or an oligosaccharideof interest comprising a galactose-β1,4-glucose moiety at its reducingend.

In a third aspect, provided is a method for the production of lactose oran oligosaccharide of interest comprising a galactose-β1,4-glucosemoiety at its reducing end by cultivating the genetically engineeredmicrobial cell in the presence of a mixed feedstock containing glucose,and recovering the oligosaccharide of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic drawing of an exemplary embodiment of agenetically engineered microbial cell according to the invention for theproduction of 2′-fucosyllactose.

FIG. 2 shows a schematic drawing of another exemplary embodiment of agenetically engineered microbial cell according to the invention for theproduction of 2′-fucosyllactose.

FIG. 3 shows a schematic drawing of a further exemplary embodiment of agenetically engineered microbial cell according to the invention for theproduction of 2′-fucosyllactose.

FIG. 4 displays growth characteristics of E. coli strains duringcultivation on glucose (A) or a mixed-monosaccharide feedstockconsisting of glucose and fructose (B) as sole carbon- and energysource.

DETAILED DESCRIPTION

According to the first aspect, provided is a genetically engineeredmicrobial cell for the production of lactose or an oligosaccharide ofinterest comprising a galactose-β1,4-glucose moiety at its reducing end,wherein said microbial cell possesses at least one glucose transporterfor translocating glucose from the culture medium into the cytoplasm ofthe microbial cell, a UDP-galactose biosynthesis pathway forintracellular biosynthesis of UDP-galactose, and at least onegalactosyltransferase that is able to galactosylate free intracellularglucose to intracellularly produce lactose.

The genetically engineered microbial cell is able to produce lactose. Incertain embodiments, the microbial cell can utilize the lactose beingproduced by itself for the production of an oligosaccharide of interestwhich bears a galactose-β1,4-glucose moiety at its reducing end. For theproduction of said oligosaccharide of interest, it is not necessary toprovide an exogenous supply of lactose to the microbial cell.

The genetically engineered microbial cell possesses at least one glucosetransporter for translocating glucose from the culture medium saidmicrobial cell is cultivated in into the cytoplasm of the microbial cellsuch that free glucose becomes available for intracellular biosynthesisof lactose.

Typically, the genetically engineered microbial cell comprises at leastone functional gene encoding said glucose transporter which is able totranslocate glucose (Glu) from the culture medium into the cell'scytoplasm.

The term “functional gene” as used herein, refers to a nucleic acidmolecule comprising a nucleotide sequence which encodes a protein orpolypeptide, and which also contains regulatory sequences operablylinked to said protein-coding nucleotide sequence such that thenucleotide sequence which encodes the protein or polypeptide can beexpressed in/by the microbial cell bearing said functional gene. Thus,when cultivated at conditions that are permissive for the expression ofthe functional gene, said functional gene is expressed, and themicrobial cell expressing said functional gene typically comprises theprotein or polypeptide that is encoded by the protein coding region ofthe functional gene. As used herein, the terms “nucleic acid” and“polynucleotide” refer to a deoxyribonucleotide or ribonucleotidepolymer in either single- or double-stranded form, and unless otherwiselimited, encompasses known analogues of natural nucleotides thathybridize to nucleic acids in a manner similar to naturally occurringnucleotides. Unless otherwise indicated, a particular nucleic acidsequence includes the complementary sequence thereof.

The term “operably linked” as used herein, shall mean a functionallinkage between a nucleic acid expression control sequence (such as apromoter, signal sequence, or array of transcription factor bindingsites) and a second nucleic acid sequence, wherein the expressioncontrol sequence affects transcription and/or translation of the nucleicacid corresponding to the second sequence. Accordingly, the term“Promoter” designates DNA sequences which usually “precede” a gene in aDNA polymer and provide a site for initiation of the transcription intomRNA. “Regulator” DNA sequences, also usually “upstream” of (i.e.,preceding) a gene in a given DNA polymer, bind proteins that determinethe frequency (or rate) of transcriptional initiation. Collectivelyreferred to as “promoter/regulator” or “control” DNA sequence, thesesequences which precede a selected gene (or series of genes) in afunctional DNA polymer cooperate to determine whether the transcription(and eventual expression) of a gene will occur. DNA sequences which“follow” a gene in a DNA polymer and provide a signal for termination ofthe transcription into mRNA are referred to as transcription“terminator” sequences.

The term “recombinant”, as used herein with reference to a bacterialhost cell indicates that the bacterial cell replicates a heterologousnucleic acid, or expresses a peptide or protein encoded by aheterologous nucleic acid (i.e., a sequence “foreign to said cell”).Recombinant cells can contain genes that are not found within the native(non-recombinant) form of the cell. Recombinant cells can also containgenes found in the native form of the cell wherein the genes aremodified and re-introduced into the cell by artificial means. The termalso encompasses cells that contain a nucleic acid endogenous to thecell that has been modified without removing the nucleic acid from thecell; such modifications include those obtained by gene replacement,site-specific mutation, and related techniques. Accordingly, a“recombinant polypeptide” is one which has been produced by arecombinant cell. A “heterologous sequence” or a “heterologous nucleicacid”, as used herein, is one that originates from a source foreign tothe particular host cell (e.g. from a different species), or, if fromthe same source, is modified from its original form. Thus, aheterologous nucleic acid operably linked to a promoter is from a sourcedifferent from that from which the promoter was derived, or, if from thesame source, is modified from its original form. The heterologoussequence may be stably introduced, e.g. by transfection, transformation,conjugation or transduction, into the genome of the host microorganismcell, wherein techniques may be applied which will depend on the hostcell the sequence is to be introduced. Various techniques are known to aperson skilled in the art and are, e.g., disclosed in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989).

Accordingly, a “genetically engineered microbial cell” is understood asa bacterial cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

Thus, the nucleic acid sequences as used in the present invention, may,e.g., be comprised in a vector which is to be stablytransformed/transfected or otherwise introduced into host microorganismcells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and to synthesize a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., supra.

The art is rich in patent and literature publications relating to“recombinant DNA” methodologies for the isolation, synthesis,purification and amplification of genetic materials for use in thetransformation of selected host organisms. Thus, it is common knowledgeto transform host organisms with “hybrid” viral or circular plasmid DNAwhich includes selected exogenous (i.e. foreign or “heterologous”) DNAsequences. The procedures known in the art first involve generation of atransformation vector by enzymatically cleaving circular viral orplasmid DNA to form linear DNA strands. Selected foreign DNA strandsusually including sequences coding for desired protein product areprepared in linear form through use of the same/similar enzymes. Thelinear viral or plasmid DNA is incubated with the foreign DNA in thepresence of ligating enzymes capable of effecting a restoration processand “hybrid” vectors are formed which include the selected exogenous DNAsegment “spliced” into the viral or circular DNA plasmid.

The term “nucleotide sequence encoding . . . ” generally refers to anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA, and generally represents the portionof a gene which encodes a certain polypeptide or protein. The termincludes, without limitation, single- and double-stranded DNA, DNA thatis a mixture of single- and double-stranded regions or single-, double-and triple-stranded regions, single- and double-stranded RNA, and RNAthat is mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded, or triple-stranded regions, or a mixture of single- anddouble-stranded regions. The term also encompasses polynucleotides thatinclude a single continuous region or discontinuous regions encoding thepolypeptide (for example, interrupted by integrated phage or aninsertion sequence or editing) together with additional regions thatalso may contain coding and/or non-coding sequences.

In an embodiment, a suitable glucose transporter is a glucosefacilitated diffusion protein. A suitable glucose facilitated fusionprotein is encoded by the glf gene of Zymomonas mobilis subsp. mobilis(strain ATCC 31821/ZM4/CP4).

In an additional and/or alternative embodiment, another suitable glucosetransporter is a glucose translocation permease. A suitable glucosetranslocation permease is encoded by the E. coli K-12 galP gene. Theglucose translocation permease is also known as galactose-protonsymporter or galactose premease, but also imports glucose across thecell membrane.

Thus, in an additional and/or alternative embodiment, the geneticallyengineered microbial cell comprises and expresses at least one genecomprising the protein coding region of the glf gene of Zymomonasmobilis subsp. mobilis (strain ATCC 31821/ZM4/CP4), the E. coli K-12galP gene or functional variants thereof.

The term “variant(s)” as used herein, refers to a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains the essential (enzymatic) properties of thereference polynucleotide or polypeptide. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to thepersons skilled in the art.

Within the scope of the present invention, also nucleicacid/polynucleotide and polypeptide polymorphic variants, alleles,mutants, and interspecies homologs are comprised by those terms, thathave an amino acid sequence that has greater than about 60% amino acidsequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequenceidentity, preferably over a region of at least about 25, 50, 100, 200,500, 1000, or more amino acids, to a polypeptide encoded by a wildtypeprotein.

Accordingly, a “functional variant” of any of the genes/proteinsdisclosed therein, is meant to designate sequence variants of thegenes/proteins still retaining the same or somewhat lesser activity ofthe gene or protein the respective fragment is derived from.

The genetically engineered microbial cell possesses an UDP-galactosebiosynthesis pathway for intracellular formation of GDP-galactose(GDP-Gal), because an efficient supply of UDP-galactose is needed forintracellular biosynthesis of lactose.

In an additional and/or alternative embodiment, UDP-galactose can beobtained from the microbial cells' own metabolism, i.e. the activity ofa phosphoglucomutase, an UTP-glucose-1-phosphate-uridyltransferase andan UDP-glucose-4-epimerase.

The intracellular supply of GDP-galactose can be improved by geneticmodifications such as an expression or overexpression of one or more ofthe genes encoding polypeptides exhibiting phosphoglucomutase activity,UDP-glucose-1-phosphate-uridyltransferase activity andUDP-glucose-4-epimerase activity, respectively.

The term “overexpression” or “overexpressed” as used herein refers to alevel of enzyme or polypeptide expression that is greater than what ismeasured in a wildtype cell of the same species as the host cell thathas not been genetically altered.

Phosphoglucomutase is an enzyme that facilitates interconversion ofglucose-1-phosphate to glucose-6-phosphate in that it on an α-D-glucosemonomer from the 1′ to the 6′ position or the 6′ to the 1′ position. Anexemplary gene encoding a suitable phosphoglucomutase is the E. coliK-12 pgm gene (GenBank: U08369.1). Thus, in an additional and/oralternative embodiment, the genetically engineered microbial cellcomprises and expresses/overexpresses a gene encoding aphosphoglucomutase, the gene preferably comprising the protein codingregion of the E. coli pgm gene or a variant thereof.

UTP-glucose-1-phosphate-uridyltransferase such as GalU or a functionalvariant thereof catalyzes the conversion of α-D-glucose-1-phosphate toUDP-glucose utilizing UTP. An exemplary gene encoding a suitableUTP-glucose-1-phosphate-uridyltransferase is the E. coli K-12 galU gene(GenBank: M98830.1). Thus, in an additional and/or alternativeembodiment, the genertically engineered microbial cell comprises andexpresses/overexpresses a gene encoding aUTP-glucose-1-phosphate-uridyltransferase, the gene preferablycomprising the protein coding region of the E. coli galU gene or avariant thereof.

UDP-glucose-4-epimerase such as GalE or a functional variant thereofcatalyzes the epimerization of UDP-glucose to UDP-galactose. Anexemplary gene encoding a UDP-glucose-4-epimerase is the E. coli K-12galE gene. Thus, in an additional and/or alternative embodiment, thegenertically engineered microbial cell comprises andexpresses/overexpresses a gene encoding an UDP-glucose-4-epimerase, thegene preferably comprising the protein coding region of the E. coli galEgene or a variant thereof.

In an additional and/or alternative embodiment, the UDP-galactosebiosynthesis pathway additionally comprises the enzymetic activity of aglucose-6-phosphate isomerase which converts fructose-6-phosphate toglucose-6-phosphate and vice versa. An exemplary gene encoding aglucose-6-phosphate isomerase is the E. coli K-12 pgi gene. Thus, in anadditional and/or alternative embodiment, the genertically engineeredmicrobial cell comprises and expresses/overexpresses a gene encoding aglucose-6-phosphate isomerase, the gene preferably comprising theprotein coding region of the E. coli pgi gene or a variant thereof.

Alternatively, UDP-galactose can be obtained by feeding galactose to themicrobial cells via the culture medium. The galactose is taken up by thecell and phosphorylated to galactose-1-phosphate which is then convertedto UDP-galactose. Genes encoding the enzymes possessing the requiredenzymatic activities are known in the literature (Groissoird et al.,“Characterization, Expression, and Mutation of the Lactococcus lactisgalPMKTE Genes, Involved in Galactose Utilization via the Leloir Pathway(2003) J. Bacteriol. 185(3) 870-878).

The genetically engineered microbial cell comprises aβ-1,4-galactosyltransferase that is able to galactosylate free glucosemonosaccharide. In an additional and/or alternative embodiment, asuitable β-1,4-galactosyltransferase is derived from Neisseriamenningitidis, from Aggregatibacter aphrophilus of from Pasteurellamultocida, preferably a β-1,4-galactosyltransferase encoded by theNeisseria menningitidis IgtB gene, by the lex-1 gene of Aggregatibacteraphrophilus or by the β-1,4-galactosyltransferase gene galTpm1141 fromPasteurella multocida (GenBank: AEC04686). Thus, in an additional and/oralternative embodiment, the genertically engineered microbial cellcomprises and expresses/overexpresses a gene encoding aβ-1,4-galactosyltransferase, the gene preferably comprising the proteincoding region of the Neisseria menningitidis IgtB gene, theAggregatibacter aphrophilus lex-1 gene, the Pasteurella multocidagalTpm1141 gene or a variant thereof.

The β-1,4-galactosyltransferase uses UDP-galactose as substrate for thetransfer of the galactose moiety to the free glucose monosaccharidethereby synthesizing a galactose-β1,4-glucose disaccharide, i.e.lactose.

In an additional and/or alternative embodiment, the geneticallyengineered microbial cell comprises at least one additionalglycosyltransferase, i. e. in addition to saidβ-1,4-galactosyltransferase.

Generally, and throughout the present disclosure, the term“glycosyltransferase activity” or “glycosyltransferase” designates andencompasses enzymes that are responsible for the biosynthesis ofdisaccharides, oligosaccharides and polysaccharides, and they catalyzethe transfer of monosaccharide moieties from an activated nucleotidemonosaccharide/sugar (the “glycosyl donor”) to a glycosyl acceptormolecule.

In a preferred embodiment, the at least one additionalglycosyltransferase is a fucosyltransferase, a sialyltransferase, aglucosaminyltransferase or a galactosyltransferase, more preferably, theat least one additional glycosyltransferase is selected from at leastone of the following: alpha-1,2-fucosyltransferase,alpha-1,3-fucosyltransferase, beta-1,3-N-acetylglucosamyltransferase,beta-1,3-galactosyltransferase, alpha-2,3-sialyltransferase,alpha-2,6-sialyltansferase, beta-1,4-galactosyltransferase orbeta-1,6-galactosyltransferase.

The enzymatic activity of the at least one additionalglycosyltransferase allows production of oligosaccharides of interestwhich comprise a galactose-β-1,4-glucose moiety at their reducing end byusing lactose as an acceptor for the activity of the additionalglycosyltransferase. Table 1 identifies the most abundant HMOs which maybe produced by the microbial cells and methods disclosed herein asoligosaccharide of interest.

TABLE 1 List of oligosaccharides of interest that can be produced byusing a genetically modified microbial cell and/or a method as describedherein. Name Abbrev. structure 2′-Fucosyllactose 2′-FLFuc(α1-2)Gal(β1-4)Glu 3-Fucosyllactose 3-FL

2′,3-Difucosyllactose DF-L

Lacto-N-triose II LNT II GlcNAc(β1-3)Gal(β1-4)Glu Lacto-N-tetraose LNTGal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glu Lacto-N-neotetraose LNnTGal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glu Lacto-N- LNFP IFuc(αβ1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glu fucopentaose I Lacto-N-LNnFP I Fuc(α1-2)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glu neofucopentaose ILacto-N- fucopentaose II LNFP II

Lacto-N- fucopentaose III LNFP III

Lacto-N- fucopentaose V LNFP V

Lacto-N- neofucopentaose V LNnFP V

Lacto-N- difucohexaose I LNDH I

Lacto-N- difucohexaose II LND

6′-Galactosyllactose 6′-GL Gal(β1-6)Gal(β1-4)Glu 3′-Galactosyllactose3′-GL Gal(β1-3)Gal(β1-4)Glu Lacto-N-hexaose LNH

Lacto-N-neohexaose LNnH

para-Lacto-N- paraLNTGal(β1-3)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glu hexaosepara-Lacto-N- paraLNnHGal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glu neohexaoseDifucosyl-lacto-N- neohexaose DF-LNnH

3′-Sialyllactose 3′-SL Neu5Ac(α2-3)Gal(β1-4)Glu 6′-Sialyllactose 6′-SLNeu5Ac(α2-6)Gal(β1-4)Glu Lacto-N- LST-aNeu5Ac(α2-3)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glu sialylpentaose a Lacto-N-sialylpentaose b LST-b

Lacto-N- LST-c Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glusialylpentaose c Fucosyl-lacto-N- sialylpentaose a F-LST-a

Fucosyl-lacto-N- sialylpentaose b F-LST-b

Fucosyl-lacto-N- sialylpentaose c F-LST-c

Disialyl-lacto-N- tetraose DS-LNT

Disialyl-lacto-N- fucopentaose DS-LNFP V

3-Fucosyl-3′- sialyllactose 3F-3′-SL

3-Fucosyl-6′- sialyllactose 3F-6′-SL

Lacto-N- neodifucohexaose I LNnDFH I

In an additional and/or alternative embodiment, the microbial cellcomprises a glucose-translocating phosphotransferase system (PtsG). Theglucose-translocating phosphotransferase system catalyzes thephosphorylation of incoming glucose concomitantly with its translocationacross the cell membrane.

The general mechanism of the Pts system is the following: a phosphorylgroup from phosphoenolpyruvate (PEP) is transferred via a signaltransduction pathway, to enzyme I (EI) which in turn transfers it to aphosphoryl carrier, the histidine protein (HPr). Phospho-HPr thentransfers the phosphoryl group to a sugar-specific permease, amembrane-bound complex known as enzyme 2 (EII), which transports thesugar to the cell. EII consists of at least three structurally distinctdomains IIA, IIB and IIC. These can either be fused together in a singlepolypeptide chain or exist as two or three interactive chains, formerlycalled enzymes II (EII) and III (EIII).

The first domain (IIA or EIIA) carries the first permease-specificphosphorylation site, a histidine which is phosphorylated byphospho-HPr. The second domain (IIB or EIIB) is phosphorylated byphospho-IIA on a cysteinyl or histidyl residue, depending on the sugartransported. Finally, the phosphoryl group is transferred from the IIBdomain to the sugar substrate concomitantly with the sugar uptakeprocessed by the IIC domain. This third domain (IIC or EIIC) forms thetranslocation channel and the specific substrate-binding site.

Thus, the PtsG system acquires exogenous glucose and providesglucose-6-phosphate in the microbial cell. Glucose-6-phosphate caneither be utilized in the UDP-galactose biosynthesis pathway and/orconverted to fructose-6-phosphate which in turn may be used forgenerating energy-rich triphosphates in the central metabolism and/or,for example, in the biosynthesis of nucleotide activated saccharidessuch as GDP-fucose.

In an additional and/or alternative embodiment, the glucokinase gene(s)of the microbial cell have been deleted or functionally inactivated suchthat the microbial cell does not possess any polypeptide havingglucokinase activity. Glucokinase (Glk)b phosphorylates free glucose atits carbon atom 6 to generate glucose-6-phosphate. In the absence ofglucokinase activity, free glucose that is translocated into themicrobial cell's cytoplasm becomes available as substrate for theβ1,4-galactosyltransferase to produce lactose, whereas theglucose-6-phosphate obtained from PtsG activity may be utilized forUDP-galactose formation or other metabolic pathways.

In an additional and/or alternative embodiment, the geneticallyengineered microbial cell comprises a fructose transporter fortranslocating fructose (Fru) from the culture medium into the microbialcell's cytoplasm. A suitable fructose transporter for uptake of freefructose is an isoform (PtsG-F) as described by Kornberg et al. PNAS 97:1808-1812 (2000)).

The internalized fructose may then be phosphorylated by a fructokinase(FrK) to provide fructose-6-phosphate (Fru-6-P). Fructose-6-phosphatemay be utilized in the UDP-galactose biosynthesis pathway and/or inother metabolic pathways such as generating energy-rich triphosphates inthe central metabolism and/or, for example, in the biosynthesis ofnucleotide activated saccharides such as GDP-fucose.

In an additional and/or alternative embodiment, the geneticallyengineered microbial cell comprises polypeptides exhibitingfructokinase-6 activity and polypeptides exhibiting6-phosphofructokinase-1 activity (FruK or phosphofructokinase) toprovide a metabolic pathway from internalized fructose viafructose-6-phosphate to fructose-1.6-bisphosphate.

In an additional and/or alternative embodiment, the geneticallyengineered microbial cell comprises a fructose-translocatingphosphotransferase system (PtsF). The fructose-translocatingphosphotransferase system catalyzes the phosphorylation of incomingfructose concomitantly with its translocation across the cell membrane.

Thus, the PtsF system acquires exogenous fructose and providesfructose-1-phosphate in the microbial cell. The PtsF system comprises amembrane-spanning protein FruA, a 1-phosphofructose kinase (FruK) and adiphosphoryl transfer protein FruB. Fructose is translocated by means ofFruA and FruB to provide fructose-1-phosphate in the cytoplasm.Fructose-1-phosphate can be further phosphorylated by aphosphofructokinase (FruK) to yield fructose-1,6-bisphosphate which inturn may be used by the microbial cell for generating energy-richtriphosphates in the central metabolism.

Another suitable PtsF System comprises LevD, LevE, LevF and LevG. LevDis the fructose-specific phosphotransferase enzyme IIA component. LevEis the fructose-specific phosphotransferase enzyme IIB component. LevFis the fructose permease IIC component, LevG is the fructose permeaseIID component. Corresponding genes levD, levE, levF and levG are—forexample known form Bacillus subtilis (strain 168). Said PtsF systemprovides fructose-1-phosphate in the cell.

In an additional and/or alternative embodiment, the geneticallyengineered microbial cell comprises at least one 1-phosphofructokinase(FruK).

In an additional and/or alternative embodiment, the geneticallyengineered microbial cell comprises a fructose-1,6-bisphosphatase(GlpX). Said fructose-1,6-bisphosphatase dephosphorylatesfructose-1,6-bisphosphate to provide fructose-6-phosphate. Thefructose-6-phosphate may be used by the microbial cell in theGDP-galactose biosynthesis pathway or in another metabolic pathway forexample, in the biosynthesis of nucleotide activated saccharides such asGDP-fucose.

Preferably, the microbial cell also comprises a deletion or functionalinactivation of its phosphofructokinase gene(s). Deletion or functionalinactivation of the phosphofructokinase gene(s) leads to a microbialcell that lacks phosphofructokinase activity such that the conversion ofFru-6-P to Fru-1,6-bisP is prevented. In E. coli, two isoforms ofphosphofructokinase are present, designated PfkA and PfkB. Thecorresponding genes are pfkA and pfkB.

In an additional and/or alternative embodiment, the geneticallyengineered microbial cell possesses a GDP-L-fucose biosynthesis pathway.In an additional and/or alternative embodiment, the GPD-fucosebiosynthesis pathway comprises a mannose-6-phosphate isomerase (ManA), aphosphomannomutase (ManB), a mannose-1-phosphate-guanylyltransferase(ManC), a GDP-mannose-4,6-dehydratase (Gmd), a GDP-L-fucose synthase(WcaG). Preferably, the microbial cell possessing a GDP-L-fucosebiosynthesis pathway also possesses a fucosyltransferase.

In an additional and/or alternative embodiment, the microbial cellcomprises an exporter protein or a permease exporting theoligosaccharide of interest from the cell, preferably a sugar effluxtransporter.

In an additional and/or alternative embodiment, the geneticallyengineered microbial cell comprises a deletion of functionalinactivation of its glucose-6-phosphate isomerase gene such that themicrobial cell lacks glucose-6-phosphate isomerase activity.Glucose-6-phosphate isomerase, in E. coli designated Pgi, convertsglucose-6-phosphate to fructose-6-phosphate. By deleting theglucose-6-phosphate gene(s) or by inactivating their expression, anyglucose-6-phosphate present in the cytoplasm of the microbial cell canbe directed towards lactose production.

In an additional and/or alternative embodiment, the microbial cell is abacterial cell selected from the group consisting of bacteria of thegenera Escherichia, Lactobacillus, Corynebacterium, Bacillus,Streptococcus, Enterococcus, Lactococcus and Clostidium, preferably abacterial cell that is selected from the group of bacterial speciesconsisting of Escherichia coli, Corynebacterium glutamicum, Clotridiumcellulolyticum, Clostridium ljungdahlii, Clostridium autoethanogenum,Clostridium acetobutylicum, Bacillus subtilis, Bacillus megaterium,Lactobacillus casei, Lactobacillus acidophilus, Lactobacillushelveticus, Lactobacillus delbrueckii, and Lactococcus lactis. Inanother embodiment, the microbial cell is an Escherichia coli. A personskilled in the art will be aware of further bacterial strains whenreading the present disclosure.

According to the second aspect, provided is the use of a geneticallyengineered microbial cell as described herein for the production oflactose or an oligosaccharide of which comprises agalactose-β1,4-glucose moiety at its reducing end. In an additionaland/or alternative embodiment, the genetically engineered microbial cellis used in a method for the production of lactose or an oligosaccharideof which comprises a galactose-β1,4-glucose moiety at its reducing end,wherein the microbial cell is cultivated in the presence of a mixedfeedstock comprising glucose and at least one additional carbon source.Said additional carbon source may be selected from the group consistingof fructose, galactose, mannose, xylose, rhamnose, glycerol, succinate,pyruvate and malate. Preferably, the mixed feedstock is a mixture ofglucose and fructose, more preferably an equimolar mixture of glucoseand fructose, most preferably comprising or consisting of hydrolyzedsucrose.

According to the third aspect, provided is a method for the productionof lactose or an oligosaccharide of interest which comprises agalactose-β1,4-glucose moiety at its reducing end, the method comprisingthe steps of:

-   -   a) providing a genetically engineered microbial cell as        described herein;    -   b) cultivating the microbial cell in a culture medium and under        conditions that are permissive for the production of said        lactose or oligosaccharide of interest, wherein the culture        medium contains a mixture of glucose and at least one additional        compound selected from the group consisting of fructose,        galactose, mannose, xylose, rhamnose, glycerol, succinate,        pyruvate and malate as main carbon source; and    -   c) recovering the lactose or oligosaccharide of interest from        the culture medium and/or the microbial cell.

In an additional and/or alternative embodiment, the oligosaccharide ofinterest is a human milk oligosaccharide selected from the groupconsisting of 2′-fucosyllactose, 3-fucosyllactose,2′,3-difucosyllactose, 3′-sialyllactose, 6′-sialyllactose,3-fucosyl-3′-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose,lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaoseIII, lacto-N-fucopentaose V, lacto-N-difucosylhexose I,lacto-N-difucosylhexaose II, lacto-N-sialylpentaose LSTa, LSTb, LSTc.

Preferably, the mixture of glucose and at least one additionalmonosaccharide is a mixed feedstock of glucose and fructose, preferablyobtained by hydrolyzation of sucrose.

In an additional and/or alternative embodiment, the microbial cell iscultivated without exogenous supply of lactose, in particular whencultivated for the production of the oligosaccharide of interest.

The present invention will be described with respect to particularembodiments and with reference to drawings, but the invention is notlimited thereto but only by the claims. Furthermore, the terms first,second and the like in the description and in the claims, are used fordistinguishing between similar elements and not necessarily fordescribing a sequence, either temporally, spatially, in ranking or inany other manner. It is to be understood that the terms so used areinterchangeable under appropriate circumstances and that the embodimentsof the invention described herein are capable of operation in othersequences than described or illustrated herein.

It is to be noticed that the term “comprising”, used in the claims,should not be interpreted as being restricted to the means listedthereafter; it does not exclude other elements or steps. It is thus tobe interpreted as specifying the presence of the stated features,integers, steps or components as referred to, but does not preclude thepresence or addition of one or more other features, integers, steps orcomponents, or groups thereof. Thus, the scope of the expression “adevice comprising means A and B” should not be limited to devicesconsisting only of components A and B. It means that with respect to thepresent invention, the only relevant components of the device are A andB.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, appearances of the phrases“in one embodiment” or “in an embodiment” in various places throughoutthis specification are not necessarily all referring to the sameembodiment, but may. Furthermore, the particular features, structures orcharacteristics may be combined in any suitable manner, as would beapparent to one of ordinary skill in the art from this disclosure, inone or more embodiments.

Similarly it should be appreciated that in the description of exemplaryembodiments of the invention, various features of the invention aresometimes grouped together in a single embodiment, figure, ordescription thereof for the purpose of streamlining the disclosure andaiding in the understanding of one or more of the various inventiveaspects. This method of disclosure, however, is not to be interpreted asreflecting an intention that the claimed invention requires morefeatures than are expressly recited in each claim. Rather, as thefollowing claims reflect, inventive aspects lie in less than allfeatures of a single foregoing disclosed embodiment. Thus, the claimsfollowing the detailed description are hereby expressly incorporatedinto this detailed description, with each claim standing on its own as aseparate embodiment of this invention.

Furthermore, while some embodiments described herein include some butnot other features included in other embodiments, combinations offeatures of different embodiments are meant to be within the scope ofthe invention, and form different embodiments, as would be understood bythose in the art. For example, in the following claims, any of theclaimed embodiments can be used in any combination.

Furthermore, some of the embodiments are described herein as a method orcombination of elements of a method that can be implemented by aprocessor of a computer system or by other means of carrying out thefunction. Thus, a processor with the necessary instructions for carryingout such a method or element of a method forms a means for carrying outthe method or element of a method.

Furthermore, an element described herein of an apparatus embodiment isan example of a means for carrying out the function performed by theelement for the purpose of carrying out the invention.

In the description and drawings provided herein, numerous specificdetails are set forth. However, it is understood that embodiments of theinvention may be practiced without these specific details. In otherinstances, well-known methods, structures and techniques have not beenshown in detail in order not to obscure an understanding of thisdescription.

The invention will now be described by a detailed description of severalembodiments of the invention. It is clear that other embodiments of theinvention can be configured according to the knowledge of personsskilled in the art without departing from the true spirit or technicalteaching of the invention, the invention being limited only by the termsof the appended claims.

In an embodiment, an E. coli strain possessing the genotype lacY⁻,lacZ⁻, fuclK⁻, wcaJ⁻ is metabolically engineered to efficiently produce2′-fucosyllactose by means of total fermentation using amixed-monosaccharide feedstock (e.g. hydrolyzed sucrose) as main carbon-and energy source. Therefore, the expression of the glucokinase gene gikand/or the glucose dehydrogenase gene gcd and/or the glucose PTSpermease gene ptsG is decreased and/or abolished. In addition, a glucosepermease gene is expressed or overexpressed in said E. coli strain.

Furthermore, at least one of the E. coli genes manA, manC, manB, gmd,wcaG, pgm, galU and galE as well as the expression of a heterologousβ-1,4-galactosyltransferase, capable to transfer galactose fromUDP-galactose on glucose, thus generating lactose, and aα-1,2-fucosyltransferase, capable to transfer fucose from GDP-fucose tolactose, thus generating 2′-fucosyllactose, are expressed/overexpressed.

In a preferred embodiment, this production strain is further engineeredby decreasing and/or diminishing the expression of thephosphofructokinase genes pfkA and/or pfkB and/or theglucose-6-phosphate dehydrogenase gene zwf and/or theglucose-6-phosphate isomerase gene pgi. This further geneticmodification allows cultivation of the thus engineered production strainon a mixed-monosaccharide feedstock (e.g. hydrolyzed sucrose) as maincarbon- and energy source, while preventing to hamper the strain'smetabolism but increasing precursor supply (glucose andfructose-6-phosphate and glucose-6-phosphate) for 2′-fucosyllactoseproduction by total fermentation.

Referring to FIG. 1, a exemplary microbial cell of the invention isschematically shown. Said microbial cell is capable of producing 2′-FLwhen cultivated on a mixed feedstock consisting of glucose (Glu) andfructose (Fru), but which mixed feedstock does not contain lactose(Lac). The microbial cell expresses polynucleotides encoding a glucosetransporter (Glf) and a fructose transporter for import of glucose andfructose into the cell respectively. As expression of the glucosekinaseGlk has been abolished by deletion or functional inactivation of the g/kgene(s), any glucose that is imported by the cell becomes available assubstrate for the β1,4-galactosyltransferase GalTpm1141 by theUDP-galactose biosynthesis pathway to intracellularly generate lactose(Lac).

Imported fructose is phosphorylated by the cell's fructose-6 kinase togenerate a intracellular Fru-6-P pool. A portion of the Fru-6-P pool isused in the “UDP-Gal biosynthesis pathway” to intracellularly synthesizeUDP-Gal which serves as Gal donor for the GalTpm1141galactosyltransferase to generate Lac. Another portion of the fru-6-Ppool is used in the “GDP-L-Fuc biosynthesis pathway” for GDP-L-fucoseproduction. Said GDP-L-fucose serves as fucose donor for the2′-fucosyltransferase WbgL. Yet a third portion of the intracellularFru-6-P pool is used for energy and biomass production in that itsfru-6-P is converted to Fru-1,6-bisP by the cells phosphofructokinasesPfkA and/or PfkB.

FIG. 2 displays schematically another a exemplary microbial cell of theinvention being capable of producing 2′-FL when cultivated on a mixedfeedstock consisting of glucose (Glu) and fructose (Fru), but whichmixed feedstock does not contain lactose (Lac). In addition to theexemplary microbial cell shown in FIG. 1, the microbial cell furthercomprises a glucose-specific Pts System (PtsG). Said PtsG system importsand phosphorylates Glu to provide Glu-6-P in the cell's cytosol. SaidGlu-6-P may be utilized by the microbial cell for generating UDP-Gal orFru-6-P. In a variant of the microbial cell (not shown), the cell's pgigene(s) encoding a glucose-6-phosphate isomerase (Pgi) is deleted. Alongwith the deletion of the glk gene, the microbial has been geneticallyengineered such that free glucose monomer as acquired by Glf becomesavailable as substrate to the β1,4-galactosyltransferase, whereas anyGlu-6-P acquired by means of PtsG becomes available for UDP-Galbiosynthesis.

FIG. 3 displays schematically another exemplary microbial cell of theinvention capable of producing 2′-FL when cultivated on a mixedfeedstock consisting of glucose (Glu) and fructose (Fru), but whichmixed feedstock does not contain lactose (Lac). In addition to theexemplary microbial cell shown in FIG. 2, the microbial cell furthercomprises a fructose-specific Pts system (PtsF). Said PtsF systemimports and phosphorlylates Fru to provide Fru-1-P. Fru-1-P isphosphorlyted by FruK to provide Fru-1,6-bisP. The microbial cellpossesses fructose-1,6-bisphosphatase (GlpX) activity. Thereby, thegenetically engineered microbial cell is metabolically engineered suchthat fructose and/or fructose-1-P can be utilized by the cell forUDP-Gal biosynthesis.

In addition, the phosphofructokinase gene(s) are deleted or functionallyinactivated such that the cell does not possess phosphofructokinase(PfkA/PfkB) activity. Deletion or functional inactivation of thephosphofructokinase genes impairs conversion of Fru-6P to Fru-1,6-P suchthat utilization of Fru-6-P for generating energy-rich triphosphates isprevented and production of 2′-FL is enhanced.

EXAMPLES Example 1—Preparation of a Mixed Monosaccharide Feedstock

A 50% (w/v) sucrose solution was prepared by dissolving 500 g of sucrosein water. The final volume of the solution was 1 litre. At a temperatureof 30° C. to 35° C. the pH was adjusted by using 96% (v/v) sulfuricacid. Afterwards, the solution was sterilized in a vertical autoclave(Systec VX-65, Linden, Germany) at 121° C. for 45 minutes. Samples weretaken before and after heat sterilization and kept frozen prior toanalysis by high performance liquid chromatography (HPLC). HPLC wascarried out using a RID-10A refractive index detector (Shimadzu,Germany) and a Waters XBridge Amide Column 3.5 μm (250×4.6 mm)(Eschborn, Germany) connected to a Shimadzu HPLC system. Isocraticelution was carried out with 30% solvent A (50% (v/v) acetonitrile indouble distilled water, 0.1% (v/v) NH4OH) and 70% solvent B (80% (v/v)acetonitrile in double distilled water, 0.1% (v/v) NH4OH) at 35° C. andat a flow rate of 1.4 mL min-1. Samples were cleared by solid phaseextraction on an ion exchange matrix (Strata ABW, Phenomenex). Tenmicroliters of the sample (dilution of 1:5) was applied to the column.Finally, the relative amount of detected sugars was determined. Asdepicted in Table 1, the sucrose conversion into the monosaccharidesglucose and fructose increased with decreasing pH values of thesolutions prior to heat treatment. Full sucrose cleavage could beobserved at pH values≤3.50 when acidification was carried out withsulfuric acid.

TABLE 1 Relative amount of sugars detected in pH adjusted 50% (w/v)sucrose solution before and after heat sterilization. The pH adjustmentwas carried out using 96% (v/v) sulfuric acid. Depicted is the percentalamount of sugars (area under the curve; AUC) detected by HPLC. Relativecomposition [%] pH Sucrose Glucose Fructose before heat sterilization3.50-7.10 100 — — after heat sterilization 7.10 (not acidified) 100 — —pH 5.50 87.55 6.30 6.15 pH 5.05 68.25 16.62 15.13 pH 3.87 13.90 45.0541.06 pH 3.50 — 51.68 48.32

Example 2—Feedstock-Dependent Growth of Various Gene Deletion Strains

The growth behavior of an E. coli BL21(DE3) strain (wild type) as wellas the mutated strains E. coli pfkA⁻ (ΔpfkA), E. coli pfkB⁻ (ΔpfkB), E.coli pfkA⁻ pfkB⁻ (ΔpfkA ΔpfkA) was compared. Genomic deletions wereperformed according to the method of Datsenko and Wanner (Proc. Natl.Acad. Sci. USA 97:6640-6645 (2000)). All strains were cultivated at 30°C. in 100 mL-shake flasks with 20 mL mineral salts medium, containing 7g·L⁻¹ NH₄H₂PO₄, 7 g·L⁻¹ K₂HPO₄, 2 g·L⁻¹ KOH, 0.3 g·L⁻¹ citric acid, 2g·L⁻¹ MgSO₄×7·H₂O and 0.015 g·L⁻¹ CaCl₂×6-H₂O, supplemented with 1mL·L⁻¹ trace element solution (54.4 g·L⁻¹ ammonium ferric citrate, 9.8g·L⁻¹ MnCl₂×4·H₂O, 1.6 g·L⁻¹ COCl₂×6·H₂O, 1 g·L⁻¹ CuCl₂×2·H₂O, 1.9 g·L⁻¹H₃BO₃, 9 g·L⁻¹ ZnSO₄×7·H₂O, 1.1 g·L⁻¹ Na₂MoO₄×2·H₂O, 1.5 g·L⁻¹ Na₂SeO₃,1.5 g·L⁻¹ NiSO₄×6·H₂O) and containing either 2% (m/v) glucose (A) or 1%(w/v) glucose/1% (w/v) fructose (B) as carbon source. Cultures wereinoculated to OD 0.1 and growth development was monitored over 26 hoursby OD₆₀₀ measurement. As shown in FIG. 2, E. coli pfkA⁻ pfkB⁻ hardlyshowed growth when glucose was provided as sole carbon- and energysource, whereas its growth was indistinguishable from the wild typestrain as well as the single deletion mutants when the mixedmonosaccharide feedstock was available.

Example 3—Total Fermentation of 2′-Fucosyllactose by an Engineered E.coli Strain During Growth on a Mixed-Monosaccharide Feedstock

An E. coli BL21 (DE3) strain exhibiting the genotype pfkA⁻, lacZ⁻,fuclK⁺, wcaJ⁻, glk⁻, gcd⁻, ptsG⁻ was further genetically engineered byoverexpressing enzymes for the de novo synthesis of GDP-Fucose (ManB,ManC, Gmd, WcaG), the 2′-fucosyltransferase gene wbgL from E. coli:O126,the sugar efflux transporter gene yberc0001_9420 from Yersiniabercovieri ATCC 43970, the glucose facilitator gene glf from Zymomonasmobilis, the β-1,4-galactosyltransferase gene galTpm1141 fromPasteurella multocida (GenBank: AEC04686) as well as the E. coli genesgalE and pgm, encoding a UDP-glucose 4-epimerase and aphosphoglucomutase, respectively. Genomic deletions were performedaccording to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci.USA 97:6640-6645 (2000)). Genomic integration of heterologous genes wasperformed by transposition. Either the EZ-Tn5TM transposase (Epicentre,USA) was used to integrate linear DNA-fragments or the hyperactiveC9-mutant of the mariner transposase Himar1 (Proc. Natl. Acad. Sci.1999, USA 96:11428-11433) was employed for transposition. The genes werecodon-optimized for expression in E. coli and prepared synthetically byGenScript cooperation.

The resulting E. coli strain was cultivated at 30° C. in a 3 L fermenter(New Brunswick, Edison, USA) starting with 1000 mL mineral salts mediumcontaining 7 g·L⁻¹ NH₄H₂PO₄, 7 g·L⁻¹ K₂HPO₄, 2 g·L⁻¹ KOH, 0.3 g·L⁻¹citric acid, 2 g·L⁻¹ MgSO₄×7·H₂O and 0.015 g·L⁻¹ CaCl₂×6-H₂O,supplemented with 1 mL·L⁻¹ trace element solution (54.4 g·L⁻¹ ammoniumferric citrate, 9.8 g·L⁻¹ MnCl₂×4-H₂O, 1.6 g·L⁻¹ COCl₂×6·H₂O, 1 g·L⁻¹CuCl₂×2·H₂O, 1.9 g·L⁻¹ H₃BO₃, 9 g·L⁻¹ ZnSO₄×7·H₂O, 1.1 g·L⁻¹Na₂MoO₄×2·H₂O, 1.5 g·L⁻¹ Na₂SeO₃, 1.5 g·L⁻¹ NiSO₄×6·H₂O) and containing2% (m/v) hydrolyzed sucrose as carbon source. Cultivation was startedwith the addition of a 2.5% (v/v) inoculum from a pre-culture grown inthe same medium. The end of the batch phase was characterized by a risein the dissolved oxygen level. A carbon feed consisting of fullyhydrolyzed sucrose, supplemented with 2 g·L⁻¹ MgSO₄×7·H₂O, 0.015 g·L⁻¹CaCl₂×6·H₂O and 1 mL·L⁻¹ trace element solution, was appliedinstantaneously after leaving the batch phase. A feeding rate of12.0-15.0 mL·L⁻¹·h⁻¹ was applied, referring to the starting volume.Aeration was maintained at 3 L·min⁻¹. Dissolved oxygen was maintained at20-30% saturation by controlling the rate of agitation. The pH wasmaintained at 6.7 by adding 25% ammonia solution. Cultivation lasted for86 hours and yielded substantial amounts of 2′-FL in the culturesupernatant.

1. A genetically engineered microbial cell for the production of lactoseor an oligosaccharide of interest which comprises agalactose-β1,4-glucose moiety at its reducing end, wherein saidmicrobial cell possesses: at least one glucose transporter fortranslocating glucose from the culture medium into the microbial cell'scytoplasm; an UDP-galactose biosynthesis pathway; and at least oneβ-1,4-galactosyltransferase being able to galactosylate free glucose tointracellularly produce lactose.
 2. The genetically engineered microbialcell according to claim 1, wherein the at least one glucose transporteris selected from the group consisting of glucose facilitated diffusionproteins and glucose translocating permeases.
 3. The geneticallyengineered microbial cell according to claim 1, wherein said microbialcell expresses or overexpresses at least one gene encoding the glucosetransporter, preferably at least one gene selected from the groupconsisting of glf, galP and functional variants thereof.
 4. Thegenetically engineered microbial cell according to claim 1, wherein saidmicrobial cell possesses a phosphoglucomutase, aUTP-glucose-1-phosphate-uridyltransferase, and a UDP-glucose4-epimerase.
 5. The genetically engineered microbial cell according toclaim 1 wherein the β-1,4-galactosyltransferase is encoded by a geneselected from the group consisting of Neisseria meningitidis IgtB,Aggregatibacter aphrophilus lex-1, Pasteurella multocida galTpm1141 andfunctional variants thereof.
 6. The genetically engineered microbialcell according to claim 1, wherein said microbial cell possesses atleast one additional glycosyltransferase, optionally aglycosyltransferase selected from the group consisting offucosyltransferases, sialyltransferases, glucosaminyltransferases andgalactosyltransferases.
 7. The genetically engineered microbial cellaccording to claim 1, wherein the microbial cell comprises aglucose-translocating phosphotransferase system.
 8. The geneticallyengineered microbial cell according to claim 1, wherein said microbialcell possesses a fructose transporter.
 9. The genetically engineeredmicrobial cell according to claim 1, wherein the microbial cellpossesses a fructose specific phosphotransferase system, and wherein thecell further comprises 1-phosphofructokinase.
 10. The geneticallyengineered microbial cell according to claim 8, wherein said microbialcell comprises fructokinase-6 activity and 6-phosphofructokinase-1activity.
 11. The genetically engineered microbial cell according toclaim 9, wherein the microbial cell comprises afructose-1,6-bisphsophatase.
 12. The genetically engineered microbialcell according to claim 9, wherein said microbial cell comprises adeletion or functional inactivation of a glucose-6-phosphate isomerase.13. The genetically engineered microbial cell according to claim 6,wherein the additional glycosyltransferase is a fucosyltransferase, andwherein said microbial cell possesses a mannose-6-phosphate isomerase, aphosphomannomutase, a mannose-1-phosphate-guanylyltransferase, aGDP-mannose-4,6-dehydratase, a GDP-L-fucose synthase.
 14. Thegenetically engineered microbial cell according to claim 1, wherein saidmicrobial cell comprises an exporter protein or a permease exporting theoligosaccharide of interest from the cell, optionally a sugar effluxtransporter.
 15. The genetically engineered microbial cell according toclaim 1, wherein said microbial cell is a bacterial cell selected fromthe group consisting of bacteria of the genera Escherichia,Lactobacillus, Corynebacterium, Bacillus, Streptococcus, Enterococcus,Lactococcus and Clostidium, optionally a bacterial cell that is selectedfrom the group of bacterial species consisting of Escherichia coli,Corynebacterium glutamicum, Clotridium cellulolyticum, Clostridiumljungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum,Bacillus subtilis, Bacillus megaterium, Lactobacillus casei,Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillusdelbrueckii, and Lactococcus lactis.
 16. A product comprising agenetically engineered microbial cell according to claim 1 forproduction of lactose or an oligosaccharide, wherein the lactose oroligosaccharide comprises a galactose-β1,4-glucose moiety at itsreducing end.
 17. A method for production of lactose or anoligosaccharide of interest which comprises a galactose-β1,4-glucosemoiety at its reducing end, the method comprising: a) providing agenetically engineered microbial cell according to claim 1; b)cultivating the microbial cell in a culture medium and under conditionsthat are permissive for production of said lactose or oligosaccharide ofinterest, wherein the culture medium contains a mixture of glucose andat least one additional compound selected from the group consisting offructose, galactose, mannose, xylose, rhamnose, glycerol, succinate,pyruvate and malate as main carbon source; and c) recovering the lactoseor oligosaccharide of interest from the culture medium and/or themicrobial cell.
 18. The method according to claim 17, wherein theoligosaccharide of interest is a human milk oligosaccharide selectedfrom the group consisting of 2′-fucosyllactose, 3-fucosyllactose,2′,3-difucosyllactose, 3′-sialyllactose, 6′-sialyllactose,3-fucosyl-3′-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose,lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaoseIII, lacto-N-fucopentaose V, lacto-N-difucosylhexose I,lacto-N-difucosylhexaose II, lacto-N-sialylpentaose LSTa, LSTb, LSTc.19. The method according to claim 17, wherein the mixture of glucose andat least one additional monosaccharide is a mixed feedstock of glucoseand fructose, optionally obtained by hydrolyzation of sucrose.
 20. Themethod according to claim 17, wherein the microbial cell is cultivatedwithout exogenous supply of lactose.